This invention relates to virus replicated in vertebrate cell cultures, which are suitable for use in vaccine production. Viral strains useful as vaccines can be adapted to vertebrate cells by serial passage and optimization of replication of the vaccine strains. Vaccine strains adapted for growth in vertebrate cells offer many advantages. The invention also relates to methods for making and using such replicated viruses such as for vaccine.
Cultured vertebrate cells have been used for virus replication and have been classified into at least three distinct groups. Primary cells, derived from normal tissue; diploid cell strains are those that have a limited, finite life span, depending on the species of origin; and continuous cell lines, cultured cells that replicate indefinitely and may be capable of growth in suspension culture. Hayflick, in Continuous Cell Lines as Substrates for Biologicals, Arlington, Va., p2 (1988).
At present most viral vaccines are produced using primary cells or diploid cell strains. Species of origin include monkeys, chicken, embryos, and hamsters. These cell cultures have certain advantages such as easy preparation using simple media and bovine sera and sensitivity to a wide range of viruses. However, primary cells suffer from disadvantages, such as contamination by various adventitious agents, variable quality and sensitivity, and difficulty in obtaining suitable tissue for cell cultivation, low virus titers and the high cost of obtaining and preparing such cell cultures. Diploid cell strains have many of the same disadvantages as well as a finite life span that limits expansion of the cells.
African green monkey kidney (Vero) cells are a continuous cell line, that is not tumorigenic, and is suitable and advantageous for human vaccine production. In fact, several licensed vaccines are currently manufactured in these cells. Vero cells, and possibly other continuous cell lines, may be suitable for dengue virus vaccine production. However, it is not known whether the antigenic characteristic of a vaccine virus strain would not be altered after passaging in a continuous cell line.
The present invention relates to methods, replicated viruses and vaccine compositions using high growth strains of dengue virus.
The invention thus provides for a method for increasing dengue viral replication in culture by passaging the desired viral strain in continuous vertebrate cells and choosing the viral strain which replicates to high titer as the seed strain. When the dengue strain is attenuated, the resulting seed strain can be suitable for production of vaccine.
Preferably, the vertebrate host cells are continuous cell cultures derived from epithelial cells or fibroblasts, as mammalian cell lines of passage number 10-250. Preferably used for vaccine production are Vero cells as a continuous line of a passage number of about 20-250, currently available and certified (e.g., by the WHO).
Dengue virus of the invention, in isolated, purified or concentrated from, preferably has an infectivity titer of about 106-109 (such as 106-107, 107 and 108-109, or any range or value therein) plaque forming units (PFU) per ml.
The present invention also provides vaccine compositions comprising at least one strain of a dengue virus of any of the four serotypes of dengue of the present invention, in inactivated or attenuated form, optionally further comprising at least one of: (a) at least one pharmaceutically acceptable carrier or diluent; (b) at least one adjuvant and/or (c) at least one therapeutic agent.
The present invention also provides a method for eliciting an immune response to at least one dengue virus strain in a mammal, which response is prophylactic or therapeutic for a dengue virus infection. The method comprises administering to the mammal a vaccine composition comprising an inactivated and/or attenuated dengue virus of the present invention. The composition is provided in an amount that is protective or therapeutic for the mammal against a dengue virus pathology caused by infection with dengue virus.
Other objects, features, advantages, utilities and embodiments of the present invention will be apparent to skilled practitioners from the following detailed description and examples relating to the present invention.